Regulation of Cytokine Expression by HIV:Potential points of therapeutic intervention. Macrophages play a critical role in the pathogenesis of HIV infection as targets for virus replication and as sources of multifunctional cytokines. Our early studies showed that normal human monocytes stimulated with the HIV-1 envelope protein, gp120, produce the HIV-modulatory cytokines, TNF-a, IL-1b, IL-6 and GM-CSF (JI 1991) and the potent vasoactive peptide, ET-1 (JI 1993). Later studies with monocyte derived macrophages (MDM) revealed that HIV-1 infection fails to induce these latter cytokines in vitro, but consistently induces M-CSF production (JI 1995), which facilitates monocyte differentiation and increases the susceptibility of MDM to HIV infection by enhancing expression of the HIV receptors, CD4 and CCR5. Viral specificity of M-CSF production in MDM was determined using a panel of single-stranded RNA viruses (HIV-1, measles [MV] & respiratory syncytial viruses [RSV], which showed that only M-tropic strains of HIV-1 caused enhanced production of M-CSF (JI 2000). Other studies revealed that primary CXCR4- and CCR5-using isolates of both HIV-1 and HIV-2 can replicate in MDM and consistently induce M-CSF. Production of the chemokines MIP-1alpha/beta and MCP-1 by infected cells varied, suggesting that chemokine production may correlate with pathogenicity while M-CSF enables survival. Studies using anti-retroviral agents (AZT and Ritonavir) confirmed that HIV-1 replication in MDM and M-CSF production are inextricably linked, such that inhibition of one leads to comcommitant inhibition of the other (ARHR 2002). Since HIV arose from cross-species transmission of SIV and progressed from a benign to highly pathogenic disease which initially targets MDM, we looked at the ability of SIV to infect human MDM and found that 75% of SIV isolates belonging to 5 different lentivirus families could infect MDM (JAIDS 2003). SIV isolates that replicated in MDM also induced production of M-CSF but, as for HIV-2, varied with regard to the profile of chemokines produced. Ongoing studies will provide insight into lentivirus components that determine human pathogenicity with the hope of identifying additional targets for therapeutic intervention. Because astrocytes surround HIV-producing cells within the brain and could influence virus replication, we studied the effect of astrocytes on HIV expression in human MDM. Co-culture of HIV-infected MDM with human astrocytes reduced HIV replication, due in part to an unidentified astrocyte secreted factor (AIDS 1999) and to expression of inducible nitric oxide synthase (iNOS) and astrocyte production of NO (Blood 1999). Our data indicate that astrocytes play a pivotal role in determining the course of neurologic HIV disease via production of HIV modulating cytokines and expression of iNOS/NO. Thus, neurologic damage observed in HIV disease may be due to prolonged, high level production of NO by astrocytes, which may reflect a host attempt to inhibit virus replication. Modulation of HIV Replication by Cytokines, Cytokine Antagonists, and Inhibitors of Virus Binding/Fusion. We have conducted studies to identify cytokines or their antagonists (anti-cytokine antibodies or soluble cytokine receptors) capable of inhibiting HIV replication in human macrophages (MO) or T cells. Subsequent to showing that HIV-infected MO produce high levels of M-CSF and MIP-1a, which enable an HIV/MO reservoir to be established, we found that antagonists to M-CSF inhibit HIV replication and reduce production of MIP-1a when added to infected MO in vitro. These M-CSF antagonists may have clinical benefit as therapies for HIV by blocking production of virus by MO, reducing chemokine recruitment of HIV susceptible cells, and preventing establishment/maintenance of HIV-infected MO reservoirs in vivo (JI 2000). The lymphokine IL-2, used for restoration of CD4+ T cells in AIDS patients, was reported to increase M-CSF in human monocytes and cause a transient burst of HIV mRNA in plasma after administration. We asked whether MO were the source of released HIV and found that exposure of MO to IL-2 prior to HIV infection in vitro leads to a dramatic decrease in virus replication, which correlates with an IL-2-induced decrease of CD4 and CCR5 expression. Production of M-CSF was not enhanced, which suggests that IL-2 may be beneficial in preventing MO infection as well as restoring T cell function (AIDS 1998). Interferon-alpha (IFN-a) species differ in their ability to inhibit HIV replication in vitro, which may correlate with the varying therapeutic effects. We found that IFN-a species were effective at inhibiting HIV-1 replication in human MO, but showed variation in T cells that paralleled HIV cellular tropism (M vs. T cell tropic). No correlation existed between antiviral and antiproliferative activity; modulation of CD4/CCR5/CXCR4 expression on MO and T cells was not observed. Subsequent studies revealed that HIV inhibition by IFN-a depends on both the cell type infected with HIV and the co-receptor used for virus entry, such that the therapeutic effect of IFN-a changes from one that is beneficial to one that is detrimental for HIV disease. We recently initiated studies to identify proteins distinct from CD4, CCR5 and CXCR4 and involved in HIV entry in order to identify novel agents that prevent HIV envelope fusion with the cell membrane. We confirmed studies showing that protein disulfide isomerase (PDI) is involved in HIV entry and have shown that PDI antagonists will block CXCR4 Env-mediated cell fusion and spread of virus infection. We also found that PDI facilitates thiol/disulfide rearrangement in gp120 during conformational change and that inhibition of this redox shuffling prevents gp41 from assuming fusogenic conformation. At the virus-cell contact site, we find gp120 induces assembly of PDI, CD4 and CXCR4 into a protein complex serving as a portal for virus entry. Our findings support the hypothesis that PDI works in concert with CD4 and CXCR4 to effectively lower the activation energy barrier hindering Env conformational rearrangement (revised; Blood). Identifying biological agents that counteract the effect of bacterial toxins on human accessory cells. Anthrax, a disease caused by the gram positive bacterium, Bacillus anthracis, poses a significant threat as an agent of biological warfare. Anthrax toxin, consisting of three distinct proteins that help the bacterium evade the immune system and kill the host during systemic infection, is a major virulence factor. The toxin consists of three proteins that act in concert and includes two enzymes, lethal factor (LF) and edema factor (EF), and a protective antigen (PA). The primary cell type infected with B.anthracis is the macrophage and the clinical syndrome is reminiscent of septic shock, which may be mediated by cytokines produced in response to LT and ET. ET has been reported to have an inhibitory effect on human monocytes, causing he disruption of cytokine networks. LT reportedly causes lysis of murine macrophages, release of high levels of NO and TNF-a, and defects in macrophage signal transduction pathways. However, studies regarding the impact of anthrax toxins are contradictory and have primarily involved the use of murine macrophages, which have biological properties that often differ significantly from human macrophages. The focus of our laboratory concerns the role of human monocytes/macrophages in infectious diseases and the impact of these diseases on cytokine networks. We propose to study the effects of the anthrax toxin proteins alone and in combination on human monocyte/macrophage function, including cytokine production. Studies with Dr. David Frucht in DMA/LCB will determine whether differences exist between murine and human macrophages under controlled conditions and whether the murine model is appropriate for pre-clinical studies for therapeutics proposed for use in the treatment of anthrax. E.coli expression vectors for the three anthrax toxin components have been obtained from Dr. John Collier. Expression and purification of anthrax proteins that are endotoxin-free is ongoing in the laboratory of Dr. Steve Kozlowski in DMA. Upon completion of this process, the above studies will be initiated. The goal is to determine the therapeutic potential of cytokines and cytokine antagonists in the treatment of B. anthracis infection. Modernization of viral safety approaches and technology for monoclonal antibodies. (Kurt Brorson) Murine hybridoma cells used in the production of monoclonal antibodies (mAbs) produce endogenous type C retrovirus particles. Regulatory agencies require a demonstration that mAbs intended for human use are free of retrovirus with an adequate margin of safety. This is usually achieved by performing small scale evaluation studies to demonstrate that the manufacturing process is capable of removing or inactivating model viruses, including a murine retrovirus. In previous reports, we demonstrated the utility and acceptability of TaqMan fluorogenic 5'nuclease Product-Enhanced Reverse Transcriptase (TM-PERT) assays for measuring type C particles in rodent cell culture samples and RT removal by laboratory-scale models of processing steps. In another collaborative study, CBER and Genentech, Inc., used TM-PERT and a genome specific Q-PCR assay to measure retrovirus expression in scale down and full production scale Chinese hamster ovary (CHO) cell cultures of four monoclonal antibodies and one recombinant protein before and after process changes. Our data support the measuring of endogenous retrovirus output when a new cell line is introduced into manufacturing or after process changes that significantly increase cell line-specific productivity or alter the metabolic state, but suggest that re-assessment of retrovirus expression after other process changes may be unnecessary. Bracketing and generic validation of robust virus removal steps was initially proposed by the by FDA in 1997 as an approach to streamline and update the viral safety assurance strategy for IND stage mAb products. These approaches have the potential to eliminate unnecessary testing and give greater flexibility to industry during process development. This flexibility is likely to spur product development, particularly by small start-up biotechnology firms and academic sponsors of clinical IND studies. We subsequently published two matrix/ bracket studies, one which examines murine retrovirus inactivation by low pH incubation and a second looking at SV40 removal by anion exchange chromatography. Both studies identified "bracketed generic" clearance conditions where the unit operation consistently achieved significant virus clearance (> 4.5 log10). A potential safety concern in biotechnology purification schemes that employ re-use of column media, often for large numbers chromatography runs, is loss of the virus removal capacity of the chromatographic purification operation over time. To define chromatography performance attributes that best predict retrovirus clearance during extended re-use of protein A media, DMA/CBER and ONDC/CDER cycled small-scale protein A sepharose fast flow columns 150-460 times using concentrates of murine hybridoma cell culture supernatants, standard low pH elution buffers and different cleaning solutions (chaotropic and NaOH based). Retrovirus LRV was measured periodically using TM-PERT, while step yield was measured using an ELISA. Under all conditions, retrovirus clearance did not decrease after extended cycling. In two studies in which the columns were cleaned with NaOH, the chromatography performance attribute that best predicted the column media lifespan was column capacity, as measured by Ab step yield and breakthrough. For media cycled 300+ times using chaotropic cleaning buffers, column performance was more stable, although small upward trends in antibody breakthrough were evident. Thus, we propose that virus removal validation studies should be performed on new media only and these quality attributes can be monitored during protein A unit operations in lieu of performing virus removal validation studies with cycled protein A media (Brorson et al., J. Chromatography A, 2003. Studies are ongoing with Genentech to examine media decay in other resin types (e.g. ion exchange resins) and protein A resins with different matrix and linkage chemistries. This project incorporates FY2002 projects 1Z01BO002004-10, 1Z01BO002005-10, and 1Z01BO002008-01.